Detection of Borrelia burgdorferi sensu stricto by reverse line blot in the joints of Dutch patients with Lyme arthritis.
(33/1307)
OBJECTIVE: To analyze the presence of Borrelia burgdorferi sensu lato in synovial samples from the knee joint of patients with Lyme arthritis by polymerase chain reaction, and to differentiate the species by reverse line blot (RLB). METHODS: Synovial fluid (SF) and synovial tissue (ST) samples were obtained from patients with Lyme arthritis (n = 4) and from patients with various other forms of arthritis (n = 9). DNA extracted from synovial samples was amplified by using, as a target, the spacer region between the 5S and 23S ribosomal RNA genes of B. burgdorferi sensu lato. Subsequently, 4 species-specific DNA probes were used in the RLB for specific hybridization. RESULTS: DNA from B. burgdorferi sensu stricto DNA was detected in the SF and ST from 3 patients with Lyme arthritis. B. burgdorferi sensu lato DNA was not detected in the synovial samples from 9 control patients. CONCLUSION: The relationship between different species of B. burgdorferi sensu lato and arthritis can be studied using direct analysis of extracted DNA from joint samples. This method can be used to study the association between particular clinical manifestations of Lyme disease and different species of B. burgdorferi sensu lato. (+info)
Selection of RAPD marker for growth of seedlings at low temperature in rice.
(34/1307)
We have developed a polymerase chain reaction (PCR)-based assay that could effectively reduce the time period required to screen and select the cold tolerance gene of rice seedlings under field conditions. The two specific random amplified polymorphic DNA (RAPD) fragments for the assay were identified on the basis of quantitative trait loci (QTL) analysis which were found to be tightly linked to cold sensitivity. The two RAPD fragments, OPT8(600) in the cold sensitivity rice cultivar 'Dular (indica)' and OPU20(1200) in the resistance rice cultivar 'Toyohatamochi (japonica)', were identified after screening 11 RAPD fragments using 2 random primers on the genomic DNAs of 'Dular' and 'Toyohatamochi'. These primers, when used in a multiplexed PCR, specifically amplified a 0.6 kb and a 1.2 kb fragment in the sensitive and resistant rice cultivars, respectively. When this assay was performed on the genomic DNAs of 16 japonica, 3 Tongil (indica/ japonica), and 2 indica rice cultivars, the primers amplified a 0.6 kb fragment in all of the cold sensitivity rice cultivars or 1.2 kb fragment in all of the resistance ones. These markers can be of potential use in the marker-assisted selection (MAS) for cold tolerance in rice seedling. As screening for resistance can now be conducted independent of the availability of low temperature, the breeding of cold tolerance cultivars can be hastened. (+info)
Genetic diversity within Lactobacillus sakei and Lactobacillus curvatus and design of PCR primers for its detection using randomly amplified polymorphic DNA.
(35/1307)
The genotypic and phenotypic diversity among isolates of the Lactobacillus curvatus/Lactobacillus graminis/Lactobacillus sakei group was evaluated by comparing RAPD data and results of biochemical tests, such as hydrolysis of arginine, D-lactate production, melibiose and xylose fermentation, and the presence of haem-dependent catalase. Analyses were applied to five type strains and to a collection of 165 isolates previously assigned to L. sakei or L. curvatus. Phenotypic and RAPD data were compared with each other and with previous DNA-DNA hybridization data. The phenotypic and genotypic separation between L. sakei, L. curvatus and L. graminis was clear, and new insights into the detailed structure within L. sakei and L. curvatus were obtained. Individual strains could be typed by RAPD and, after the elimination of similar or identical isolates, two sub-groups in both L. curvatus and L. sakei were defined. The presence or absence of catalase activity further distinguished the two L. curvatus sub-groups. By cloning and sequencing specific RAPD products, pairs of PCR primers were developed that can be used to specifically detect L. curvatus, L. sakei and each of the L. sakei sub-groups. (+info)
Molecular identification of Lactobacillus hilgardii and genetic relatedness with Lactobacillus brevis.
(36/1307)
Conventional phenotypic methods lead to misidentification of the lactic acid bacteria Lactobacillus hilgardii and Lactobacillus brevis. Random amplified polymorphic DNA (RAPD) and repetitive element PCR (REP-PCR) techniques were developed for a molecular study of these two species. The taxonomic relationships were confirmed by analysis of the ribosomal operon. Amplified DNA fragments were chosen to isolate L. hilgardii-specific probes. In addition to rapid molecular methods for identification of L. hilgardii, these results convincingly proved that some strains first identified as L. brevis must be reclassified as L. hilgardii. The data clearly showed that these molecular methods are more efficient than phenotypic or biochemical studies for bacterial identification at the species level. (+info)
Phenotypic and genotypic characterization of non-starter lactic acid bacteria in mature cheddar cheese.
(37/1307)
Non-starter lactic acid bacteria were isolated from 14 premium-quality and 3 sensorially defective mature Irish Cheddar cheeses, obtained from six manufacturers. From countable plates of Lactobacillus-selective agar, 20 single isolated colonies were randomly picked per cheese. All 331 viable isolates were biochemically characterized as mesophilic (i.e., group II) Lactobacillus spp. Phenotypically, the isolates comprised 96.4% L. paracasei, 2.1% L. plantarum, 0.3% L. curvatus, 0.3% L. brevis, and 0.9% unidentified species. Randomly amplified polymorphic DNA (RAPD) analysis was used to rapidly identify the dominant strain groups in nine cheeses from three of the factories, and through clustering by the unweighted pair group method with arithmetic averages, an average of seven strains were found per cheese. In general, strains isolated from cheese produced at the same factory clustered together. The majority of isolates associated with premium-quality cheese grouped together and apart from clusters of strains from defective-quality cheese. No correlation was found between the isomer of lactate produced and RAPD profiles, although isolates which did not ferment ribose clustered together. The phenotypic and genotypic methods employed were validated with a selection of 31 type and reference strains of mesophilic Lactobacillus spp. commonly found in Cheddar cheese. RAPD analysis was found to be a useful and rapid method for identifying isolates to the species level. The low homology exhibited between RAPD banding profiles for cheese isolates and collection strains demonstrated the heterogeneity of the L. paracasei complex. (+info)
Distribution and evolution of the xylanase genes xynA and xynB and their homologues in strains of Butyrivibrio fibrisolvens.
(38/1307)
The ruminal bacterium Butyrivibrio fibrisolvens is being engineered by the introduction of heterologous xylanase genes in an attempt to improve the utilization of plant material in ruminants. However, relatively little is known about the diversity and distribution of the native xylanase genes in strains of B. fibrisolvens. In order to identify the most appropriate hosts for such modifications, the xylanase genotypes of 28 strains from the three 16S ribosomal DNA (rDNA) subgroups of Butyrivibrio fibrisolvens have been investigated. Only 4 of the 20 strains from 16S rDNA group 2 contained homologues of the strain Bu49 xynA gene. However, these four xynA-containing strains, and two other group 2 strains, contained members of a second xylanase gene family clearly related to xynA (subfamily I). Homologues of xynB, a second previously described xylanase gene from B. fibrisolvens, were identified only in three of the seven group 1 strains and not in the group 2 and 3 strains. However, six of the group 1 strains contained one or more members of the two subfamilies of homologues of xynA. The distribution of genes and the nucleotide sequence relationships between the members of the two xynA subfamilies are consistent with the progenitor of all strains of B. fibrisolvens having contained a xynA subfamily I gene. Since many xylanolytic strains of B. fibrisolvens did not contain members of either of the xynA subfamilies or of the xynB family, at least one additional xylanase gene family remains to be identified in B. fibrisolvens. (+info)
Outbreak of mupirocin-resistant staphylococci in a hospital in Warsaw, Poland, due to plasmid transmission and clonal spread of several strains.
(39/1307)
An outbreak of mupirocin-resistant (MuR) staphylococci was investigated in two wards of a large hospital in Warsaw, Poland. Fifty-three MuR isolates of Staphylococcus aureus, S. epidermidis, S. haemolyticus, S. xylosus, and S. capitis were identified over a 17-month survey which was carried out after introduction of the drug for the treatment of skin infections. The isolates were collected from patients with infections, environmental samples, and carriers; they constituted 19.5% of all staphylococcal isolates identified in the two wards during that time. Almost all the MuR isolates were also resistant to methicillin (methicillin-resistant S. aureus and methicillin-resistant coagulase-negative staphylococci). Seven of the outbreak isolates expressed a low-level-resistance phenotype (MuL), whereas the remaining majority of isolates were found to be highly resistant to mupirocin (MuH). The mupA gene, responsible for the MuH phenotype, has been assigned to three different polymorphic loci among the strains in the collection analyzed. The predominant polymorph, polymorph I (characterized by a mupA-containing EcoRI DNA fragment of about 16 kb), was located on a specific plasmid which was widely distributed among the entire staphylococcal population. All MuR S. aureus isolates were found to represent a single epidemic strain, which was clonally disseminated in both wards. The S. epidermidis population was much more diverse; however, at least four clusters of closely related isolates were identified, which suggested that some strains of this species were also clonally spread in the hospital environment. Six isolates of S. epidermidis were demonstrated to express the MuL and MuH resistance mechanisms simultaneously, and this is the first identification of such dual MuR phenotype-bearing strains. The outbreak was attributed to a high level and inappropriate use of mupirocin, and as a result the dermatological formulation of the drug has been removed from the hospital formulary. (+info)
Skin carriage of acinetobacters in Hong Kong.
(40/1307)
We studied the carriage of Acinetobacter spp. at five superficial sites in 79 patients from two hospitals, in 133 healthy controls from the community (medical students and new nurses), and in 198 student nurses in different classes. A total of 431 isolates from 364 positive sites of 201 subjects and 124 blood culture isolates (1997 to 1998) were genospeciated by amplified ribosomal DNA restriction analysis. Genospecies 3 was the most common species. The carriage rate of student nurses (42 of 131) was significantly lower than that of new nurses from the community (25 of 38) (chi-square test, P = 0.0004; odds ratio [OR], 4.08; 95% confidence limits, 1.78 to 9.41) but not significantly different (P = 0.1) from that of patients in the same hospital (20 of 42). Genospecies from blood cultures and subjects (acute patients and student nurses) from Prince of Wales Hospital were similar to one another but different from subjects from the community or from another hospital (chi-square test, P < 0.0001). Half of the subjects who were positive at at least two sites had different genospecies. Of the 28 sites examined, 68% showed strain variation among isolates of the same genospecies by random amplified polymorphic DNA analysis. Half of the 106 subjects who had samples taken again within 6 weeks or 6 months later were positive only once. In the 17 subjects who were positive on at least two occasions, each occasion yielded different genospecies in 13 subjects. Our results indicate that skin carriage in the majority of healthy subjects is characterized by low density, variation in genospecies and strains, short-term duration, and the typicality of a given locality. (+info)